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A primordial tRNA modification required for the evolution of life?

2001; Springer Nature; Volume: 20; Issue: 1 Linguagem: Inglês

10.1093/emboj/20.1.231

1460-2075

Glenn R. Björk,

Parasitic Infections and Diagnostics

Article15 January 2001free access A primordial tRNA modification required for the evolution of life? Glenn R. Björk Corresponding Author Glenn R. Björk Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Kerstin Jacobsson Kerstin Jacobsson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Kristina Nilsson Kristina Nilsson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Marcus J.O. Johansson Marcus J.O. Johansson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Anders S. Byström Anders S. Byström Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Olof P. Persson Olof P. Persson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Glenn R. Björk Corresponding Author Glenn R. Björk Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Kerstin Jacobsson Kerstin Jacobsson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Kristina Nilsson Kristina Nilsson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Marcus J.O. Johansson Marcus J.O. Johansson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Anders S. Byström Anders S. Byström Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Olof P. Persson Olof P. Persson Department of Microbiology, Umeå University, S-90187 Umeå, Sweden Search for more papers by this author Author Information Glenn R. Björk 1, Kerstin Jacobsson1, Kristina Nilsson1, Marcus J.O. Johansson1, Anders S. Byström1 and Olof P. Persson1 1Department of Microbiology, Umeå University, S-90187 Umeå, Sweden *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:231-239https://doi.org/10.1093/emboj/20.1.231 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m1G37), which prevents frameshifting and is present adjacent to and 3′ of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m1G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m1G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m1G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m1G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m1G37)methyltrans ferase is part of the minimal set of gene products required for life. Introduction Following the chemical evolution of life, a condensation period may have occurred resulting in a population of primitive cells (progenotes) with a rudimentary translation apparatus (Woese, 1998). This translation apparatus was simple and presumably not able to maintain the reading frame for a long distance. Whereas most missense errors are not fatal for the activity and stability of a protein, frameshift errors are detrimental for the synthesis of proteins, which are required for a more evolved and efficient organism. Thus, an early feature to evolve would have been structures that were important for maintenance of the reading frame. Such structures may have been a prerequisite for the emergence of the three domains Archaea, Bacteria and Eukarya. Since tRNA is the primary decoding molecule in all organisms, some tRNA features, e.g. the presence of some early evolved modified nucleosides, may still play a pivotal role in reading frame maintenance and thus be found in tRNA of present-day organisms from all three domains. Transfer RNA from all organisms contains modified nucleosides, which are derivatives of the four canonical nucleosides: adenosine (A), guanosine (G), uridine (U) and cytidine (C). At present, 79 different modified nucleosides have been characterized in tRNAs from various organisms (Rozenski et al., 1999). Not only are two positions in the tRNA, i.e. the wobble position (position 34) and the position adjacent to and 3′ of the anticodon (position 37), frequently modified, but a plethora of various modified nucleosides are also found in these two positions (Auffinger and Westhof, 1998; Björk, 1998). Of all the modified nucleosides present in tRNA, only eight are present in the same position and in the same subpopulation of tRNA in organisms from all domains (Björk, 1986), suggesting that they may have been present in the tRNA of the primitive organisms existing before the emergence of the three domains Archaea, Bacteria and Eukarya (Björk, 1986; Cermakian and Cedergren, 1998). If a convergent evolution has not occurred, derivatives of the ancestor genes, whose products catalyse the synthesis of these conserved modified nucleosides, will be present in the organisms of today and these modified nucleosides may still have the same function in all organisms. One of these eight conserved modified nucleosides is 1-methylguanosine (m1G37), which is present in position 37 in tRNAs specific for leucine (CUN codons, N being any of the four major nucleosides), proline (CCN) and one of the arginine tRNAs (CGG) from all three domains (Björk, 1998). In fact, among the >500 sequenced tRNAs, only three tRNA species [tRNAHisGUG from HeLa cells, tRNALeuIAG from Caenorhabditis elegans and tRNAArgPCU (P being pseudouridine Ψ) from Ascaris suum] have an unmodified G in position 37 (Sprinzl et al., 1999). Not only is m1G37 present in all organisms, including the organism with the smallest sequenced genome (Mycoplasma genitalium; Fraser et al., 1995), but it is also present in mitochondria and chloroplasts. Although the 'universal' code is used in most of the present-day organisms, the codes in Mycoplasma spp and in organelles are somewhat different (Watanabe and Osawa, 1995). tRNAs in organelles and in Mycoplasma spp are much less modified than cytosolic tRNAs (Sprinzl et al., 1999) and they often have a structure that is distinct from those of cytosolic tRNAs (Dirheimer et al., 1995). Still, the presence of m1G37 prevails in tRNA, even in Mycoplasma and in organelles (Björk, 1998). Interestingly, the presence of this conserved modified nucleoside has been shown to prevent frameshifting (Björk et al., 1989; Hagervall et al., 1993). Therefore, the conservation of m1G37 may fulfil the above suggestion that a structure important for the early evolving capacity to maintain the reading frame may still exist in present-day organisms. If indeed a gene whose product was responsible for the formation of m1G37 in tRNA was present in the population of primitive organisms existing before the three domains emerged, sequence similarities of the orthologues may still exist. Here this question is addressed by characterizing the orthologues that catalyse the synthesis of m1G in position 37 of the tRNA from the three domains. The analysis of the DNA sequence of the smallest genome so far for an autonomously replicating organism (Fraser et al., 1995) initiated a discussion of what is the minimal set of genes required for cellular life. Theoretical considerations suggested that out of the 468 predicted protein-coding genes in M.genitalium, 256 were part of the minimal gene set (Mushegian and Koonin, 1996), and this suggestion was supported recently by transposon mutagenesis (Hutchison et al., 1999). In Salmonella typhimurium and Escherichia coli, the gene trmD, which encodes the enzyme catalysing the formation of m1G37 in tRNA, is the third gene in a four-cistron operon transcribed in the following order: rpsP (encoding ribosomal protein S16), rimM (RimM), trmD [tRNA(m1G37) methyltransferase; the TrmD protein] and rplS (ribosomal protein L19) (Byström et al., 1983). In M.genitalium, the operon organization is conserved except that the rimM gene is not present. Since the first and last genes of the M.genitalium operon encode proteins that are essential in E.coli (encoding S16 and L19, respectively; Persson et al., 1995), no transposon would be expected in the trmD gene even if it is not essential, since such an insertion would have a polarity effect on the expression of the downstream gene encoding the essential ribosomal protein L19. If the presence of m1G37 in tRNA is essential for growth, the trmD gene would be part of the minimal gene set. Therefore, it would contribute to our understanding of what is required as a minimal genome for free-living organisms if we know the influence that m1G37 in tRNA has on the growth capacity of organisms. Here we characterize the trmD orthologues from organisms belonging to each of the three domains Archaea, Bacteria and Eucarya. We show that sequence similarities still exist between these orthologues, consistent with the suggestion that an ancestor gene encoding an enzyme with similar catalytic activity may have been present before the divergence occurred. We further show that in organisms belonging to either Bacteria or Eucarya, lack of m1G37 in tRNA severely impairs growth to a similar degree. Thus, this conserved modified nucleoside plays a pivotal role in the survival of the organisms in a competitive environment, and consequently belongs to the minimal set of genes required for cellular life. Since the presence of the conserved m1G37 prevents frameshifting (Björk et al., 1989; Hagervall et al., 1993), our results are also consistent with the suggestion that one of the early improvements to the translational apparatus was the ability to maintain the reading frame, thus allowing the synthesis of long peptides necessary for the evolution of present-day organisms. Results Lack of m1G in tRNA from S.typhimurium severely impairs growth The trmD gene, which encodes tRNA(m1G37)methyl transferase (the TrmD peptide), is part of a tetra-cistronic operon transcribed in the following gene order: rpsP(S16)–rimM–trmD–rplS (L19) (Byström et al., 1983). Since deletions or nonsense codons in the trmD gene may also influence the expression of the downstream essential gene rplS (Persson et al., 1995), we isolated missense mutations that abolished the activity of the tRNA(m1G37)methyltransferase and did not have a polarity effect on the expression of the downstream rplS gene. To obtain such non-polar mutations in the trmD gene, we devised a selection procedure for strains deficient in m1G37, as described in Materials and methods. All mutations were shown to be in the trmD gene by complementation analysis using various plasmids (see Materials and methods). The various mutations were scattered all over the trmD gene (Table I). Note that no mutation was obtained that altered a sense codon to a nonsense codon, although the specificity of the mutagen used allows such mutations to occur. This fact suggests that such mutations may be lethal due to a polarity effect on the expression of rplS in addition to the phenotype mediated by the m1G37 deficiency. Table 1. m1G37 levels in tRNA and growth deficiency of S.typhimurium trmD mutants trmD alleles trmD+ trmD23 trmD27/31 trmD30 trmD28 trmD25 trmD26 trmD3/24 trmD33 trmD29 trmD32 Amino acid substitution P58L/L94F S88L G117S G117N G117Q S165L P184L G199R G214D W217D m1G/Ψa 0.12 0.006 <0.005 0.005 <0.005 0.014 0.005 0.047 0.006 <0.005 0.040b Relative colony size atc 22°C 1 (1.4 mm)d No col.e 0.3 0.6 No col.e 1.1 −e 1.1 <0.1 1.1 −e 30°C 1 (3 mm)d 0.4 0.7 0.7 0.2 0.8 <0.1 0.8 0.5 0.9 <0.1 37°C 1 (1.8 mm)d 0.4 0.7 0.6 <0.1 0.7 0.1 0.9 0.4 0.1–0.7 <0.1 42.5°C 1 (2.2 mm)d <0.1 <0.1 0.3 No col.e 0.4 0.4 0.5 0.3 <0.1 0.2–0.4 44°C 1 (2.5 mm)d No col.e No col.e 0.2 No col.e 0.3 0.3 0.3 0.1 <0.1 0.3 a The level of mG was determined by HPLC in bulk tRNA prepared from cells grown in a rich medium (LB) at 37°C and is expressed as the absorbance of mG relative to that of pseudouridine (Ψ) at 254 nm. b This level is overestimated, since Gm was trailing into the region where mG migrated. c The growth of the various mutants at the indicated temperatures was determined by single-cell outstreak on TYS plates. The relative colony size was determined as the average size of ∼5 colonies in relation to the size of the wild type. Thus, <0.1 indicates that the growth of the mutant was 90% reduction in growth at 37°C (Table I). However, the reduction in growth became much more severe at a higher temperature (trmD23 trmD27 and trmD29). Since some amino acid substitutions in the TrmD peptide influence the substrate specificity (Li and Björk, 1999), the various mutant forms of the TrmD peptide may have different substrate specificities, resulting in various levels of m1G37 in different tRNA species and, therefore, different phenotypes. The m1G37 deficiency may also induce a temperature sensitivity of tRNA(s), resulting in a more pronounced growth defect. At less optimal temperatures, the growth was much more impaired (Table I). We conclude that the absence of m1G37 severely impairs the growth of S.typhimurium. Characterization of the trmD orthologue of Methanoccocus vannielii and Methanococcus jannaschii Plasmids harbouring non-bacterial genes lacking bacterial promoters and translational signals may be expressed in bacteria, albeit at low levels. However, even a low expression of a tRNA-modifying enzyme may be enough to complement a phenotype induced by an undermodified tRNA. To obtain the trmD orthologue from an Archaeon, we introduced a plasmid bank containing chromosomal fragments from M.vannielii into the temperature-sensitive S.typhimurium strain GT5337 (trmD27, purF2085). A plasmid, pUMV4, containing a 1.3 kb M.vannielii chromosomal insert rendered the temperature-sensitive strain GT5337 able to grow at high temperature (Table II). The plasmids also increased the level of m1G in the tRNA. Thus, the 1.3 kb HindIII fragment contains the trmD orthologue of the Archaeon M.vannielii. Table 2. Complementation of the S.typhimurium trmD27 mutation by the trmD orthologues from M.vannieliia and S.cerevisiaea S.typhimurium strains Relative m1G levelb Growth ability atc m1G/Ψ % of wt 30°C 37°C 42.5°C GT3670 (trmD+) 0.13 100 1.0 1.0 1.0 GT5337 (trmD27) <0.005 <4 0.5 0.5 <0.1 GT5055 (trmD27/pUMV5a) <0.005 <4 0.2 0.2 <0.1 GT5054 (trmD27/pUMV4a) 0.053 41 0.6 0.8 0.4 GT5058 (trmD27/pUSC1a) 0.072 60 0.7 0.8 0.4 a Plasmids pUMV4 and pUMV5 have a 1.3 and 1.5 kb chromosomal insert, respectively, from M.vannielii, and the pUSC1 plasmid has an insert from S.cerevisiae. b The level of mG was determined as described in Materials and methods and is expressed as the ratio mG/Ψ and as a percentage of the level in the wild-type strain. Bacteria were grown at 37°C. c Bacteria were grown at the indicated temperatures on TYS plates, or TYS plates containing carbenicillin for the plasmid-containing strains. The relative colony size was determined as described in Table I. The colony size of the wild-type strain GT3670 was 1.3, 2.2 and 2.2 mm at 30, 37 and 42.4°C, respectively. The insert was partially sequenced and compared with the sequence of M.jannaschii. A 493 nucleotide sequence in the middle of the 1.3 kb insert was 68.8% similar to the MJ883 open reading frame (ORF) of M.jannaschii. We conclude that the MJ883 ORF of M.jannaschii is its trmD orthologue. The M.jannaschii TrmD orthologue showed 7% identity and 16% similarity to the E.coli TrmD protein. Characterization of the yeast trmD orthologue The ORF YHR070W encodes a protein of 499 amino acids with unknown function. This protein is 17% identical and 33% similar to the protein encoded by the MJ883 ORF of M.jannaschii. Thus, YHR070W is a potential yeast trmD orthologue. We cloned this gene into vector pYES2, which resulted in plasmid pUSC1. This plasmid complemented the temperature-sensitive growth of S.typhimurium strain GT5337 and raised the level of m1G in the tRNA to ∼40% of the wild-type level (Table II). The increased level of m1G was thus similar to that induced by the 1.3 kb HindIII fragment of M.vannielii. We conclude that YHR070W is the yeast trmD orthologue and we denote this gene TRM5 in accordance with the nomenclature for other yeast genes encoding tRNA methyltransferases. Deletion of TRM5 in Saccharomyces cerevisiae severely impairs growth Having identified the TRM5 gene as the potential structural gene for the tRNA(m1G37)methyltransferase of S.cerevisiae, we deleted it by inserting the HIS3 gene between codons 19 and 463 of the 499 amino acid TRM5 gene in the diploid strain GBY1, generating strain GBY3 (TRM5/trm5::HIS3). Upon sporulation of the diploid strain GBY3, His+ and His− segregants were obtained at a ratio of 2:2. Moreover, extremely slow growing segregants co-segregated with the His+ phenotype, linking the slow-growing phenotype with the trm5::HIS3 allele. All slow-growing segregants from several tetrads analysed showed the same growth defect, ruling out that this residual growth was caused by an accumulation of some extragenic suppressors. Figure 1 shows that the TRM5 segregants (His−) formed large colonies after 2 days of incubation at 30°C, whereas the His+ segregants (trm5::HIS3) needed 9 days of incubation to grow to small colonies. Thus, a deletion of the TRM5 gene severely impaired growth. Figure 1.Growth of the S.cerevisiae congenic pairs TRM5 and trm5::HIS3, and TRM5 and trm5::kanMX4 (see key to strain at the bottom of the figure) on rich plates after 2 and 9 days, respectively, of incubation at 30°C. After 2 days of incubation, there were no visible colonies of the trm5 mutant. Download figure Download PowerPoint A previously constructed mutant of the YHR070W ORF contained a substitution of a kanMX4 gene between the start and the stop codons. Analysis of this mutant showed that this gene was essential (Saccharomyces Genome Deletion Project); thus we found it necessary to clarify this discrepancy. The diploid strain BY4743 (TRM5/trm5::kanMX4; obtained from Research Genetics) was sporulated, and fast- and slow-growing segregants were obtained in a 2:2 ratio. All slow-growing segregants from several tetrads showed the same growth defect, suggesting, as above, that an extragenic suppressor did not cause this residual growth. The growth of the haploid deletion strain at 30°C was impaired to a degree similar to that of the deletion strain (trm5::HIS3) constructed by us (Figure 1). Thus, with both constructs, our analysis showed that the TRM5 gene is not essential, although a deletion of it severely impairs growth to a degree similar to that observed for the S.typhimurium trmD mutants. The yeast tRNA(m1G37)methyltransferase, encoded by the TRM5 gene, catalyses the transfer of a methyl group if a G or an inosine is present in position 37 We next determined the m1G content in bulk tRNA from the haploid TRM5 and trm5::HIS3 strains. Table III shows that in the mutant strain the m1G level was reduced by only 21% and lacked yW (the nucleoside of the Y-base present in yeast tRNAPhe; Figure 2). It is known that eukaryotic tRNA species also have m1G in position 9 (Sprinzl et al., 1999), the formation of which is most probably catalysed by an enzyme distinct from tRNA(m1G37)methyltrans ferase. Unlike in E.coli, tRNAHisGUG, tRNAAspIGC and tRNALeuUAG from yeast also contain m1G37. Since the tRNA(m1G37) methyltransferase from E.coli methylates only tRNA specific for leucine (decoding CUN codons), proline (CCN) and arginine (CGG), the yeast Trm5p enzyme may also methylate the same subset of tRNAs exclusively. These considerations might explain the modest decrease of m1G in bulk tRNA of the mutant. To clarify the specificity of the Trm5p enzyme in yeast, we purified various tRNA species by hybridization to matrix-bound oligonucleotides complementary to the 3′ side of the tRNA from wild-type and mutant. The purified tRNA samples, which should only contain one tRNA species, were then analysed, and the modification pattern was determined by HPLC. The results are shown in Table IV. Figure 2.Analysis of modified tRNA nucleosides from wild-type S.cerevisiae TRM5 (upper panel) and the trm5::HIS3 mutant (lower panel). Only the portion of the chromatogram between retention times 47 and 82 min is shown. Abbreviations: t6A, N6-threonylcarbamoyladenosine; Ar, 2′-O-ribosyladenosine; m6A, N6-methyladenosine; yW, wybutosine (nucleoside of the Y-base); i6A, N6-isopentenyladenosine. Download figure Download PowerPoint Table 3. Analysis of m1G content in S.cerevisiae TRM5 and the trm5::HIS3 mutant Strains Genotype No. of analyses m1G/Ψa % of wt yWb i6Ab GBY6,7 TRM5 12 0.33 ± 0.04 100 + + GBY8,9 trm5::HIS3 6 0.265 ± 0.003 79 − + aThe level of m1G was determined as described in Materials and methods and is expressed as the m1G/Ψ ratio with the range in the determinations indicated. bThe presence of yW and i6A was determined by HPLC analysis as shown in Figure 2. + indicates the presence and − the absence of the indicated modified nucleoside. Table 4. Content of modified nucleosides in various purified tRNA species in S.cerevisiae TRM5 and trm5::HIS3 strains Modified nucleoside Mutant/wild typea AlaIGC AspGUC LeuUAA LeuUAG HisGUG ProNGG ArgCCG PheGmAA Ψ 1.1 1.1 1.3 1.4 1.1 1.0 1.0 1.1 ncm5U –b – – – – 0.91 – – m1A – – 3.7c 0.97 – 1.0 – 1.0 m5C – 1.0 1.1 0.83 1.3 1.0 1.4 1.1 Cm – – 1.0 – – 1.0 – 0.96 m7G – – – – – (incl. in I) – (incl. in I) Id 0.82 1.27 2.1 1.90 1.2 0.97 0.96 1.0 m5U 1.0 1.3 1.1 1.1 1.1 0.98 0.90 0.99 Gm – – 1.4 0.78 1.5 – – 1.4 m1I <0.1 – – – – – – – m1G 1.0 <0.04 <0.1 <0.08 <0.06 0.48 <0.07 – ac4C – – 1.3 1.1 – – – – m2G – – 1.0 1.1 – – 1.6 1.0 m22G 1.0 – 1.0 1.0 – – – 1.0 Am – – – – 1.0 – – – yW – – – – – – – <0.1 a The numbers given are the ratios of the various modified nucleosides in the indicated tRNA isoacceptor isolated from the trm5::HIS3 mutant and TRM5 wild type. The underlined modified nucleoside, which was used as internal standard, is, according to the sequence, present once in the tRNA species. b – denotes that this modified nucleoside is, according to the sequence, not present in the wild-type tRNA species and, therefore, not expected to be present in the analysis of a particular tRNA. The absence of such a modified nucleoside demonstrates the purity of the tRNA analysed. c This high ratio depends on a low level of mA in the wild-type tRNAUAA. The level of mA in the trm5::HIS3 mutant was similar to that for all other tRNA species with mA. d Inosine was found in all samples, regardless of whether the tRNA should contain I or not. This is most probably caused by chemical deamination of A to I in tRNA during the purification procedure. The tRNALeuUAG and tRNAArgCCG were completely devoid of m1G37 in the trm5::HIS3 mutant. The level of m1G in tRNAProncm5UGG, which contains m1G37 and m1G9, was reduced to 50% of the wild-type level. These results show that the Trm5p enzyme methylates G37 in the same subset of tRNAs as the bacterial enzyme, but does not methylate G9. In addition, the yeast enzyme also methylates tRNAHisGUG, tRNAAspQUC and tRNALeuUAA (Table IV). Thus, the yeast Trm5p and the bacterial TrmD methylate the subset of tRNAs that have G37 and G36. In addition, the yeast enzyme also methylates tRNA species that have a G37, but also have a C36 (tRNAAspQUC) or an A36 (tRNALeuUAA). The tRNAAlaIGC contains m1G9 and m1I37 (I, inosine) instead of m1G37. The synthesis of m1I37 occurs in two enzymatic steps: the formation of I37 by the Tad1p enzyme followed by a methylation step to form m1I37, catalysed by an unknown methyltransferase (Grosjean et al., 1996; Gerber et al., 1998). Table IV shows that tRNA from both the mutant and the wild type contains m1G, consistent with our suggestion that Trm5p does not mediate the formation of m1G9. However, the mutant lacks m1I37, showing that Trm5p catalyses the transfer of the methyl group to I37, thereby synthesizing m1I37. The tricyclic nucleoside yW (Y-base nucleoside) is one of the most complex modified nucleosides characterized so far, and is found exclusively in tRNAPheGmAA of eukaryotic organisms, including yeast. Its presence in yeast tRNAPheGmAA, whose three-dimensional structure was the first to be established for a tRNA (Kim et al., 1974; Robertus et al., 1974), evoked much attention regarding not only its function but also its use as a natural fluorescent probe in molecular analysis of the mechanism of translation. The first step in the synthesis of this complex modified nucleoside in yeast is the formation of m1G37 (Droogmans and Grosjean, 1987). Table IV and Figure 2 show that tRNAPheGmAA from the trm5::HIS3 mutant was devoid of yW, suggesting that the tRNA(m1G37)methyl transferase encoded by the TRM5 gene mediates the first step in the synthesis of yW. This is the first gene to be identified whose product is involved in the synthesis of the Y-base. We conclude that the tRNA(m1G37)methyltransferase encoded by the TRM5 gene in S.cerevisiae catalyses the synthesis of m1G37 in the subset of tRNAs [Leu(CUN), Pro(CCN) and Arg(CGG)] for which m1G37 is conserved. This fact demonstrates that the yeast Trm5p enzyme has a similar and overlapping substrate specificity to that of the bacterial TrmD enzyme. In addition, it also mediates the formation of m1I37 in tRNAAspQUC and yW in tRNAPheGmAA. Unlike the bacterial TrmD enzyme, it is not dependent on the nature of the nucleoside 5′ of the target nucleoside (position 36 can be at least A, G and C). Since the Trm5p from yeast also methylates I37, it does not require an amino group in position 2, as found in G, of the target purine. However, an amino group at position 6, as in adenine, seems to inhibit Trm5p, since several tRNAs in yeast have an unmodified A in position 37. Our results establish furthermore that there is only one tRNA(m1G37) methyltransferase in yeast, since all of the tRNAs analysed that have G37 were shown to be the substrate of the Trm5p enzyme (Table IV). Discussion We have identified experimentally the orthologues encoding the tRNA(m1G37)methyltransferase from the three domains Archaea, Bacteria and Eukarya (Table II and Figure 3). The ubiquitous presence of m1G37 in the same subset of present-day tRNAs suggests that this modification may be pivotal for the survival and evolution of different organisms in a competitive environment. Results presented here support this view, since a lack of m1G37 severely impaired the growth of both a bacterium and a eukaryote (Table I and Figure 1). The growth defect in mutants lacking m1G37 in combination with the high conservation of this modified nucleoside suggest that a gene (trmD orthologue) responsible for the synthesis of m1G37 must be part of a minimal gene set. Sequence similarities between these orthologues suggest that organisms present before the three domains emerged possessed an ancestor gene responsible for the formation of m1G37 in their tRNA. Furthermore, our results suggest that the yeast tRNA(m1G37)methyltransferase also catalyses a step in the formation of the tricyclic modified nucleoside yW37 (nucleoside of the Y-base) and m1I37 (Figure 2 and Table IV). U